1. Field of the Invention
The present invention relates to a method for pre-treating a specimen and a method for analyzing a specimen.
2. Description of the Related Art
The importance of analysis of proteins which are gene products present in living organisms has come under close scrutiny with recent development in genome analysis.
The importance of expression and functional analysis of proteins has been pointed out, and development of an analysis method is being advanced.
Such a method basically includes a combination of (1) separation and purification by two-dimensional electrophoresis and high-performance liquid chromatography (HPLC) and (2) detection and analysis, such as radiometric analysis, optical analysis, mass spectrometry, and the like.
The basis of protein analysis techniques is called “proteome analysis” which aims at analyzing proteins derived from genes and actually functioning in living organisms and at investigating the functions of cells and the causes of diseases.
A typical analysis method includes the following:
(1) extraction of proteins from a biological tissue or cells to be examined;
(2) separation of the proteins by two-dimensional electrophoresis;
(3) analysis of the proteins or segments thereof by mass spectrometry such as a MALDI method (Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry: MALDI-TOFMS); and
(4) identification of the proteins using a database such as Genome Project.
For example, proteins involved in recurrence and metastasis of a cancer are being clarified by the proteome analysis, and the results thereof start to appear.
On the other hand, a technique relating to a pre-treatment step in the series of proteome analysis, for example, a pre-treatment technique for analyzing only target cells from a tissue sample containing normal cells and abnormal cells in a development stage of a cancer or the like, becomes very important.
Such a pre-treatment is generally performed by a technique of extracting target cells from a sample.
For example, U.S. Pat. No. 6,184,973 discloses an apparatus using microdissection.
The target cells are collected from a tissue sample using this apparatus according to the following procedures:
First, a glass slide on which a tissue sample has been fixed is placed at a center of a stage, and a sampling cap is moved using a cap transfer portion.
The glass slide is moved by manually operating the stage while the tissue sample is observed through an observation optical system of an inverted optical microscope to find the target cells in the tissue sample.
Next, the glass slide is positioned so that the target cells are at the center of a field of view.
Then, a laser beam is applied through the sampling cap, and only the target cells in the tissue sample are bonded using a thermoplastic portion of the sampling cap.
The sampling cap is separated from the glass slide to collect the target cells together with the sampling cap. Another known microdissection apparatus is of a type in which a target portion is cut out with a laser beam.
Apart from this, Japanese Patent Laid-Open No. 2003-098060 discloses a method of selectively collecting a target portion using a probe microscope.
The method disclosed in this document includes the following steps: coating a target sample with a coating material, forming an image of the coated sample using a probe microscope, curetting the target portion with a probe of the probe microscope, treating the target portion exposed by curettage, and selectively collecting a substance contained in the target portion.
Further, US 2004/0219588 discloses a method of analyzing only a target portion in a tissue sample by treating the target portion without physically taking out target cells from the tissue sample.
Specifically, in this method, an analytical reagent is adhered to a portion to be analyzed (the order of several 100 microns) by an ink jet method so that the portion is analyzed directly by a microscopic observation method or laser desorption/ionization mass spectrometry.
In the apparatus disclosed in U.S. Pat. No. 6,184,973, it is necessary to position a target cell at the center of the field of view while manually operating the stage for each of cells scattered at a plurality of points in the same tissue sample. In addition, it is necessary to apply a laser beam after the positioning operation, thereby requiring much labor and time for the work.
The technique using the probe microscope disclosed in Japanese Patent Laid-Open No. 2003-098060 includes coating the target portion with the coating agent, exposing the target portion by curetting with the probe, and analyzing the target portion. Therefore, reproducibility and analysis accuracy may be decreased.
Further, the method disclosed in US 2004/0219588 includes applying an analysis reagent to a specified region of a biological sample using an ink jet method and analyzing the specified region, and thus has limitations on the analysis reagent and analysis method.